The present application relates to a method for designing a heat-resistant tyrosine-dependent short-chain dehydrogenase/reductase and a heat-resistant tyrosine-dependent short-chain dehydrogenase/reductase. In particular, the present application relates to, for example, a method for obtaining a heat-resistant mutant tyrosine-dependent short-chain dehydrogenase/reductase by changing a protein structure.
Enzymes are in vivo catalysts which facilitate many reactions related to life support under a moderate condition in a living body smoothly. The enzymes undergo metabolic turnover in the living body, and are produced in the living body as necessary, so as to perform catalytic functions.
At present, technologies to use the enzymes outside the living body have become commercially practical or have been studied toward commercialization. The technologies to use the enzymes have been developed in various technical fields, for example, production of useful materials, production of energy-related materials, measurement or analysis, environmental conservation, and medical care. In relatively recent years, technologies related to an enzyme cell which is one type of fuel cells (refer to Japanese Unexamined Patent Application Publication No. 2004-71559, for example), an enzyme electrode, an enzyme sensor (a sensor to measure chemical materials through the use of enzyme reactions), and the like have also been proposed.
Regarding the technology of fuel cell, developments proceed from fuel cells in the related art, in which combustible materials, e.g., methanol and hydrogen, are used as fuels, to fuel cells in which compounds, e.g., glucose, with a higher level of safety are used as fuels. However, metal catalysts, e.g., platinum, effectively act on highly reactive compounds, e.g., methanol and hydrogen, having relatively simple structures, whereas the catalyst efficiencies of metal catalysts are very low with respect to compounds, e.g., glucose, exhibiting a low level of danger or toxicity and having low reactivity. Consequently, as described above, enzyme cells, in which enzymes are used as catalysts, have been proposed. The enzymes have high catalytic performance, exhibit excellent substrate specificity and stereoselectivity and, therefore, efficiently catalyze reactions of compounds, e.g., glucose, having low reactivity, so that a safe fuel cell can be realized by incorporating the enzyme in a electrode of a fuel cell.
In the case where the enzyme is used outside the living body, it is important that the activity of the enzyme is high and the enzyme reaction rate is high. Furthermore, it is also necessary that the stability against changes in environment is high and the durability of the activity is high. However, the chemical body of the enzyme is a protein and, therefore, there are problems in that denaturation due to heat, pH, or the like occurs easily, and the stability outside the living body is low as compared with the other chemical catalysts, e.g., metal catalysts. In order to address the problems, Japanese Unexamined Patent Application Publication No. 2007-143493 and Japanese Unexamined Patent Application Publication No. 2008-48703 disclose a mutant enzyme (diaphorase), wherein the levels of the activity and the heat resistance are raised to at least predetermined levels by changing artificially a base sequence of a gene coding a protein, so as to produce a mutant protein.